U.S. patent application number 17/610421 was filed with the patent office on 2022-06-30 for high yield lactic acid production using mixed cultures.
This patent application is currently assigned to NATURE'S PRINCIPLES B.V.. The applicant listed for this patent is NATURE'S PRINCIPLES B.V.. Invention is credited to Maximilienne Toetie ALLAART, Robbert KLEEREBEZEM, Julius Laurens ROMBOUTS, Gerben STOUTEN, Marinus Cornelius Maria VAN LOOSDRECHT, David Gregory WEISSBRODT.
Application Number | 20220205001 17/610421 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220205001 |
Kind Code |
A1 |
ROMBOUTS; Julius Laurens ;
et al. |
June 30, 2022 |
HIGH YIELD LACTIC ACID PRODUCTION USING MIXED CULTURES
Abstract
The present invention is in the field of a method of producing
lactic acid in high yield in a sequencing batch reactor. Therein
glucose may be used as feedstock for bacteria, which ferment the
glucose into lactic acid. The reactor is operated under at least
partly defined non-axenic conditions and in a cyclic mode.
Inventors: |
ROMBOUTS; Julius Laurens;
(Delft, NL) ; VAN LOOSDRECHT; Marinus Cornelius
Maria; (Delft, NL) ; KLEEREBEZEM; Robbert;
(Delft, NL) ; WEISSBRODT; David Gregory; (Delft,
NL) ; STOUTEN; Gerben; (Delft, NL) ; ALLAART;
Maximilienne Toetie; (Delft, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATURE'S PRINCIPLES B.V. |
Den Haag |
|
NL |
|
|
Assignee: |
NATURE'S PRINCIPLES B.V.
Den Haag
NL
|
Appl. No.: |
17/610421 |
Filed: |
May 8, 2020 |
PCT Filed: |
May 8, 2020 |
PCT NO: |
PCT/EP2020/062951 |
371 Date: |
November 10, 2021 |
International
Class: |
C12P 7/56 20060101
C12P007/56; C12M 1/34 20060101 C12M001/34; C12M 1/02 20060101
C12M001/02; C12M 1/00 20060101 C12M001/00; C12N 1/20 20060101
C12N001/20 |
Foreign Application Data
Date |
Code |
Application Number |
May 10, 2019 |
NL |
2023113 |
Claims
1. A method of producing lactic acid in a sequencing batch reactor
comprising adaptively cycling at least once between (i) a reaction
phase, (ii) an effluent phase, and (iii) a feed phase, in the
reaction phase (ia1) maintaining the pH at a predetermined level
between 5.6 and 8.5, (ia2) maintaining the temperature at
30-80.degree. C., (ia3) stirring the reaction phase, (ib) adding a
base when the pH is below the predetermined level until the pH is
at or above the predetermined level, (ic1) allowing fermentation to
continue during a fermentation time until fermentation reaches a
stationary phase wherein the pH is substantially constant during at
least 10 minutes, (ic2) when fermentation has reached the
stationary phase removing part of the effluent to the effluent
phase, and (ic3) adding feed to the reaction phase, in the effluent
phase (iia) removing at least part of the lactic acid, and (iib)
preferably providing a remainder of the effluent phase to the feed
phase, in the feed phase (iiia) providing an aqueous feed mixture
comprising >10 g/l of a saccharide comprising compound, wherein
the saccharide compound is selected from glucose, sucrose,
fructose, galactose, lactose, disaccharides, oligo saccharides,
poly saccharides, starch, inulin, or a combination thereof, and
>1 g peptide/100 g saccharide compound, wherein the peptide
concentration in the feed phase is between 1-30 g peptide/100 g
saccharide, adding, at the start of the reaction phase, a mixed
starting culture capable of fermenting saccharide into lactic acid,
and wherein a biomass hydraulic retention time is controlled
between 4 h-144 h.
2. The method according to claim 1, wherein microbial biomass is
cycled at least once between the reaction phase, the effluent
phase, and the feed phase.
3. The method according to claim 1, wherein the feed phase
comprises >80 g/l saccharide compound.
4. The method according to claim 1, wherein the peptide
concentration in the feed phase is between 2-20 g peptide/100 g
saccharide.
5. The method according to claim 1, wherein the peptide is selected
from monopeptides, dipeptides, tripeptides, tetrapeptides, or is
provided as microbial biomass, and combinations thereof, and/or
wherein at least a part of the biomass is recycled.
6. The method according to claim 1, wherein a reactor size is
50-1000 m.sup.3.
7. The method according to claim 1, wherein the reactor is a
sequencing batch reactor or a sequencing fed batch rector.
8. The method according to claim 1, wherein no sterilization and/or
no inoculation is carried out and/or no live yeast is present in
the feed phase stock.
9. The method according to claim 1, wherein the feed phase
comprises vitamin B and/or metabolic precursor thereof.
10. The method according to claim 9, wherein the vitamin B is
selected from vitamin B1 (thiamin), vitamin B2 (riboflavin),
vitamin B3 (niacin, nicotinamide, nicotinamide riboside), vitamin
B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7
(biotin), vitamin B12 (cobalamin), a salt of said vitamins, and
combinations thereof.
11. The method according to claim 9, wherein the metabolic
precursor is selected from metabolic precursors for coenzyme in
catabolism of sugar, cofactor FAD, cofactor FMN, coenzyme NAD,
coenzyme NADP, coenzyme A, a metabolic coenzyme, a fatty acid
metabolism coenzyme, an amino acid metabolism coenzyme, and
combinations thereof.
12. The method according to claim 1, wherein a culture titer of
lactic acid of >40 g/l is maintained.
13. The method according to claim 1, wherein a magnesium (cation)
concentration in the feed phase is 0.1-5 g/l, and/or wherein a
calcium (cation) concentration in the feed phase is >1.5 mg Ca/g
saccharide.
14. The method according to claim 1, wherein the mixed starting
culture is enriched to L-lactic producing microorganisms.
15. The method according to claim 1, wherein a biomass hydraulic
retention time is controlled, between 18-96 hours.
16. The method according to claim 1, wherein the reaction phase
comprises >10% Streptococci.
17. The method according to claim 1, wherein a hydraulic retention
time (HRT) is from 1-8 days.
18. The method according to claim 1, wherein a base is selected
from hydroxides, oxides, ammonia, and combinations thereof.
19. The method according to claim 1 wherein a pH is maintained at
7.0.+-.0.5, a temperature at 30-55.degree. C., a peptide amount at
>2 g/100 g saccharide, a vitamin B at >0.1 g/l, and wherein
>98% L-lactic acid is produced (on a mol lactate/mol saccharide
comprising compound basis).
20. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of a method of
producing lactic acid in high yield in a sequencing batch reactor.
Therein glucose or other sugars may be used as feedstock for
bacteria, which ferment the glucose into lactic acid. The reactor
is at least partly operated under defined conditions and in a
cyclic mode.
BACKGROUND OF THE INVENTION
[0002] Lactic acid (CH3CH(OH)CO2H) is a simple organic chemical
compound that can be used in many applications. It is a chiral
molecule and it occurs as L- or D-lactic acid. It is noted that
lactic acid is highly soluble.
[0003] Lactic acid fermentation is performed on an industrial scale
by lactic acid bacteria (Lactobacillus species), which convert
simple carbohydrates such as glucose, sucrose, or galactose to
lactic acid, or by chemical synthesis. Lactic acid producing
bacteria can produce two moles of lactate from one mole of glucose,
or one mole of lactate from one mole of glucose as well as carbon
dioxide and acetic acid/ethanol, which latter is not preferred.
[0004] Typically, lactic acid fermentation is performed under
rather strict conditions. First of all, a relatively pure culture
is used, such as Lactobacillus delbrueckii. The aqueous solution in
which fermentation is performed is typically partly neutralized,
such as with lime. Typically, a pH of about 4.5-5.0 is used. The
fermentation temperature is >50.degree. C. as production is
often negligible up to 45.degree. C. For fermentation typically a
fermentor and seed reactor are used. In view of the pure culture
sterilization of the feedstock and equipment used, e.g. the
fermentor, is required. The yield of lactic acid is rather high,
such as >0.9 lactic acid/g glucose (yield on carbon of 90%), the
productivity is good (e.g. >5 g/l*h) and the titer relatively
high (>150 g/l lactic acid).
[0005] These prior art methods are however costly. A pure culture
may attribute to some 15% of production costs. The inoculum costs
some 3%, and energy consumption some 4%. The fermentor is
relatively large (a few thousand cubic meter), the seed reactor
also has a significant volume (a few hundred cubic meters), and the
need to be made from a relatively expensive material, such as
stainless steel, making investments relatively high. As mentioned,
proper sterilization is required, resulting in a down-time of the
fermentor of some 20%, and high consumption of energy typically
resulting in CO2 production. In unlucky cases a phage infection may
occur, resulting in the loss of a batch of feedstock and the need
to obtain a phage resistant strain, which can delay the production
process. Quite often reactors and parts thereof need to be cleaned,
which contributes to costs as well.
[0006] Some documents recite lactic acid production. Akao et al. In
Water Research, Elsevier, Amsterdam, Vol. 41, No. 8, Mar. 23, 2007,
p. 1774-1780 recites a method for producing L-lactate by
semi-continuous fermentation of garbage without sterile conditions.
Tang Jialing et al., in Waste Management, Elsevier New York Vol.
52, Mar. 31, 2016, p. 278-285 recites a method for producing lactic
acid in a sequence batch fermentation of a garbage feedstock.
Background art can be found in Liang et al., Waste management, Vol.
45, Nov. 1, 2015, p. 51-56, Zhang et al., in Bioresource
Technology, Elsevier Amsterdam, Vol. 99, No. 4, Mar. 1, 2008, p.
855-862, and US 2011/210001 A1.
[0007] Lactic acid is a precursor for polylactic acid (PLA), which
can be atactic or syndiotactic, and which polymers are
biodegradable polyesters. Lactic acid may also be used as a food
conservative, in cosmetics, and as a pharmaceutical component. The
market size for lactic acid is huge (>5 M /year) and growing.
Therefore, there is a need for an efficient method of producing
lactic acid.
[0008] The present invention therefore relates to an improved
method of producing lactic acid by fermentation, which solves one
or more of the above problems and drawbacks of the prior art,
providing reliable results, without jeopardizing functionality and
advantages.
SUMMARY OF THE INVENTION
[0009] It is an object of the invention to overcome one or more
limitations of the methods and devices of the prior art and at the
very least to provide an alternative thereto. In a first aspect the
present invention relates to a method of producing lactic acid in a
sequencing batch reactor comprising adaptively cycling (see e.g.
FIG. 1b) at least once between (i) a reaction phase, (ii) an
effluent phase, and (iii) a feed phase, preferably 2-5000 times
adaptive cycling, more preferably 4-1000, such as 5-1000 times.
Each phase is considered to relate to a period of time. By coupling
base dosing to substrate consumption and by closely monitoring a
base consumption an adaptive cycle can be ended almost immediately
when completed, consumption of substrate (e.g. glycose) is as fast
as possible, resulting in high time-averaged volumetric rates.
Typically, 1-20 adaptive cycles per day are possible, such as 2-10
adaptive cycles. Operation of the reactor can be continued for long
periods of time. Maintenance can be performed 1-4 times per year,
or less. The present method uses a mixed culture capable of
fermenting saccharide into lactic acid, which is added, such as to
the reactor. Compared to prior art methods as described above the
present method is considered to be >20% cheaper in terms of unit
production costs of lactic acid. Some reasons thereto are that no
sterilization is needed, no pure culture, such as a Lactobacillus
delbrueckii culture, is required, no inoculum reactor and seed
reactor are needed, the size of the present reactor can be a factor
smaller and still having a similar production rate, the present
method provides a high start biomass enabling a high productivity,
such as >6 g/l*h, the possibility of reusing biomass as protein
and vitamin source therewith reducing peptide and vitamin source
consumption, the (indirect) production of CO.sub.2 is reduced
significantly, scheduled down-time is low as no sterilization is
required, and unscheduled down time, such as due to phage
infection, is very unlikely due to the mixed culture comprising
multiple strains and species. The biomass may be recycled,
especially as it may be still intact, and therewith contributing to
an increase in productivity and yield. In addition, the throughput
is higher. The downside is that somewhat lower yields (70% versus
90%) are obtained, which may be due to more biomass and possible
byproduct, and in general there is no direct control over D/L ratio
lactic acid; however, under specific conditions the latter can be
resolved. Surprisingly at a pH of about 7 no detectable D-lactic
acid (<0.1%) is formed and >99.9% L-lactic acid.
[0010] Important differences between the present invention and
background art are that in the prior art the impact of the presence
or absence of proteins has not been specifically addressed as
enhancing the lactic acid yield and productivity. Only the amount
of carbohydrate and protein are quantified. Also, the presence or
absence of B vitamins in the feedstock is not mentioned. And most
importantly, none of the references demonstrate an "adaptive
cycling" process. These are considered as the most unique parts of
the invention, with the other factors enhancing the effectiveness
of the invention.
[0011] The present method comprises a reaction phase wherein (ia1)
maintaining the pH at a predetermined level between 5.6 and 8.5,
preferably between 5.9 and 8.0, more preferably between 6.4 and
7.5, such as at 7.0, which is compared to prior art methods
relatively high, (ia2) maintaining the temperature at 30-80.degree.
C., preferably at 32-72.degree. C., more preferably at
35-63.degree. C., even more preferably at 37-60.degree. C., such as
38-50.degree. C., e.g. 40-45.degree. C., which preferred range is
compared to prior art methods relatively low, (ia3) stirring the
reaction phase, such as at 1-600 rpm, (ib) adding a base when the
pH is below the predetermined level until the pH is at or above the
predetermined level. The pH can be measured directly with a simple
pH sensor. (ic1) Allowing fermentation to continue during a
fermentation time until fermentation reaches a stationary phase,
which stationary phase can be determined as then fermentation
substantially stops, and as a consequence the pH remains more or
less constant, such as wherein the pH is substantially constant
during at least 10 minutes, and no further base need to be added.
So e.g. by monitoring an amount of base added over time, the
stationary phase can be determined adequately, albeit with a small
delay in time. (ic2) When fermentation has reached the stationary
phase removing part of the effluent to the effluent phase, and
(ic3) adding feed to the reaction phase, therewith largely or fully
replenishing the reaction phase. The feed can be added subsequently
to removing effluent from the reaction phase, during reaction,
before reaction, and combinations thereof. In the effluent phase
(iia) at least part of the lactic acid is removed, and (iib)
preferably a remainder of the effluent phase is provided to the
feed phase, preferably wherein 30-70% of broth is provided to the
feed phase, such as 40-60% of broth. The lactic acid may be remover
through an outlet towards a vessel or the like, it may be
precipitated in the reactor, such as by adding Ca2+, it may be
separated over a membrane, and combinations thereof. So, a part of
the broth may be recycled. In an alternative, or in addition,
lactic acid may be removed from the reaction phase, such as by
in-situ separation. In the feed phase (iiia) an aqueous feed
mixture is provided comprising >10 g/l of a saccharide
comprising compound, wherein the saccharide compound is selected
from glucose, sucrose, fructose, galactose, lactose, disaccharides,
oligo saccharides, poly saccharides, such as with 3-100
saccharides, starch, inulin, preferably wherein the saccharide
comprising compound is a single compound, such as solely glucose,
and >1 g peptide/100 g saccharide compound, or a combination
thereof, wherein the peptide concentration in the feed phase is
between 1-30 g peptide/100 g saccharide. It has been found that in
addition to a saccharide as basic feed compound also peptides are
required in order to obtain sufficient yield. Without the amount of
peptide mentioned the yield drops to some 10%, much lower than the
present 70% typically obtained. When starting the at least one
adaptive cycle a mixed starting culture is added, at the start of
the reaction phase, which is capable of fermenting saccharide into
lactic acid, such as added into the reactor. A biomass hydraulic
retention time is controlled between 4 h-144 h, such as between 1-8
days. It is noted that the solid retention time and hydraulic
retention time may be substantially the same, especially when
continuous mixing is applied during volume exchange, such as during
effluent removal. The HRT is considered to depend on cycling time,
which is controlled by the adaptive cycling.
[0012] In a second aspect the present invention relates to an
apparatus for performing a method according to the invention, which
may be a relatively cheap apparatus, such as a plastic or concrete
apparatus, comprising a feed container, the feed container having a
volume of 1-50 m.sup.3, the feed container being in fluidic contact
with a reactor over a feed valve, the reactor having a pH-sensor in
fluidic contact with the aqueous solution of the reactor, a
reservoir comprising a base and a reservoir valve and optionally a
pump, the reactor having a volume of 10-100 m.sup.3, the reactor
being in fluidic contact with an effluent container over an
effluent valve, the effluent container having a volume of 1-50
m.sup.3, and the effluent container being in fluidic contact with
the feed container, each container and reactor having a mixer for
rotating an aqueous phase with 1-600 rpm, the effluent container
having an outlet for removing lactic acid, and the feed container
having an input for replenishing feed, and a controller adapted (a)
to open the reservoir valve when the pH drops below a predetermined
level and to close said reservoir valve when the pH has reached
said predetermined level again, (b) to open the feed valve when the
fermentation reaches a stationary phase and to add feed, (c) to
open the feed valve when the effluent has been partly removed and
to add feed to replenish the reactor, and (d) to open the effluent
valve when effluent is removed, such as when the liquid in the
reactor has reached a predetermined volume.
[0013] The present invention provides a solution to one or more of
the above-mentioned problems and overcomes drawbacks of the prior
art.
[0014] Advantages of the present description are detailed
throughout the description.
DETAILED DESCRIPTION OF THE INVENTION
[0015] In an exemplary embodiment of the present method biomass may
be adaptive cycled at least once between the reaction phase, the
effluent phase, and the feed phase. It has been found that biomass
not only contributes to fermentation of saccharide into lactic
acid, but also provides peptides, therewith improving the yield and
throughput.
[0016] In an exemplary embodiment of the present method the feed
phase may comprise >80 g/l saccharide compound, such as 100-200
g/l.
[0017] In an exemplary embodiment of the present method the peptide
concentration in the feed phase is between 1-40 g peptide/100 g
saccharide, preferably between 2-30 g/100 g, more preferably
between 5-15 g/100 g.
[0018] In an exemplary embodiment of the present method the peptide
may be selected from monopeptides, dipeptides, tripeptides,
tetrapeptides, or is provided as microbial biomass, and
combinations thereof. In an example of biomass also peptides in
yeast extract, or whey, or by-product of biological origin may be
used. And further the biomass in the present system may be
recycled, providing peptides, as well as vitamins or precursors
thereof.
[0019] In an exemplary embodiment of the present method a reactor
size may be 50-1000 m.sup.3, such as 100-300 m.sup.3. Such is much
smaller than prior art reactors, therewith significantly reducing
energy consumption in the production process, such as die to
agitation (stirring) of the reaction phase.
[0020] In an exemplary embodiment of the present method the reactor
may be a sequencing batch reactor or a sequencing fed batch rector.
The present reactor may relate to a reactor comprising at least one
of a zone of an apparatus, a phase of operation of an apparatus,
and a sub-reactor. The sequencing in combination with adaptive
cycling provides a quick start up, high throughput, and good
adaptability of the system to potential varying
characteristics.
[0021] In an exemplary embodiment of the present method no
sterilization needs to be carried out.
[0022] In an exemplary embodiment of the present method no
inoculation needs to be carried out.
[0023] In an exemplary embodiment of the present method no live
yeast is present in the feed phase stock.
[0024] The above relate to a significant reduction in costs of
operation, simplified mode of operation, reduced down-time, and
reduced risk of infections.
[0025] In an exemplary embodiment of the present method the feed
phase may comprise a vitamin B and/or metabolic precursor thereof,
preferably 10.sup.-2-10 mg vitamin B/g saccharide and/or precursor,
preferably 2*10.sup.-2-5 mg/g vitamin B and/or precursor, such as
0.05-1 mg/g. It has been found that these vitamin and precursors
improve the yield significantly.
[0026] In an exemplary embodiment of the present method the vitamin
B may be selected from vitamin B1 (thiamine), vitamin B2
(riboflavin), vitamin B3 (niacin, nicotinamide, nicotinamide
riboside), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine),
vitamin B7 (biotin), vitamin B12 (cobalamin), a salt thereof, such
as a phosphate salt, and combinations thereof.
[0027] In an exemplary embodiment of the present method the
precursor may be selected from metabolic precursors for coenzyme in
catabolism of sugar, co-factor FAD, co-factor FMN, coenzyme NAD,
coenzyme NADP, coenzyme A, a metabolic coenzyme, a fatty acid
metabolism coenzyme, an amino acid metabolism coenzyme, and
combinations thereof.
[0028] In an exemplary embodiment of the present method a culture
titer of lactic acid of >40 g/l may be maintained, typically
>50 g/l, such as >100 g/l, that is high titers are
obtainable.
[0029] In an exemplary embodiment of the present method a magnesium
(cation) concentration in the feed phase may be 0.1-5 g/l, such as
0.2-2 g/l.
[0030] In an exemplary embodiment of the present method a calcium
(cation) concentration in the feed phase may be >1.5 mg Ca/g
saccharide. So, for 10-200 g saccharide/l more than 1.5-300 mg Ca/L
may be provided.
[0031] The magnesium and calcium, typically provided as Mg2+ ion
and Ca2+ ion, are found to contribute to fermentation.
[0032] In an exemplary embodiment of the present method the mixed
culture may be enriched, such as by increasing an amount of
saccharide compound, e.g. to >100 g/l, such as >200 g/l.
[0033] In an exemplary embodiment of the present method a biomass
retention time may be controlled, such as between 1-8 days.
[0034] In an exemplary embodiment of the present method the
reaction phase comprises >10% Streptococcus, such as >50%
Streptococcus, which can be determined by a semi-quantitative
method like fluorescent in situ hybridization, or with any other
method, such as with PCR.
[0035] In an exemplary embodiment of the present method a hydraulic
retention time (HRT) may be from 1-8 days, preferably between 18-96
hours, such as 24-48 hours.
[0036] In an exemplary embodiment of the present method a base may
be selected from hydroxides, oxides, ammonia, and combinations
thereof, preferably comprising Ca.sup.2+, Na.sup.+, or Mg.sup.2+,
and combinations thereof.
[0037] In an exemplary embodiment of the present method a pH may be
maintained at 7.0.+-.0.5, a temperature at 30-55.degree. C., a
peptide amount at >2 g/100 g saccharide, a vitamin B at >0.1
g/l, and wherein >98% L-lactic acid is produced, such as
>99.9% L-lactic acid (on a mol lactate/mol saccharide comprising
compound basis). This is rather surprising that virtually no
D-lactic acid is produced, and substantially only L-lactic
acid.
[0038] The invention will hereafter be further elucidated through
the following examples which are exemplary and explanatory of
nature and are not intended to be considered limiting of the
invention. To the person skilled in the art it may be clear that
many variants, being obvious or not, may be conceivable falling
within the scope of protection, defined by the present claims.
SUMMARY OF THE FIGURES
[0039] FIGS. 1a-b, 2, and 3a-b and 4 show some details of the
present invention.
DETAILED DESCRIPTION OF FIGURES
[0040] In FIG. 1a a schematic representation of the present
adaptive cycle is shown. A feed phase is provided with a
saccharide, and typically mixed. The feed is provided, in an
adaptive cyclic mode, to a reaction phase, wherein microorganisms
produce lactic acid from the saccharide. In the reaction phase the
pH is measured. Upon decrease of the pH a source of base is
activated, and base is added until the pH reaches a predetermined
level. Then the addition of base is stopped, for the time being.
The effluent of the reaction phase is transferred to an effluent
phase, and the lactic acid is removed. A remainder of the effluent
phase is fed back to the feed phase.
[0041] Initially base is added to the reaction phase, in order to
compensate the pH for the lactic acid being formed. The amount of
base over time decreases, until a plateau is reached. Then addition
of base is stopped, as no further lactic acid is formed. The
reactor volume as function of time/phase of operation is shown.
Phases in a single adaptive cycle and theoretical associated base
dosage profiles of the sequencing batch reactor used to perform
adaptive base adaptive cycling. The adaptive cycle length was
controlled by imposing a maximum base constant time, after which a
new adaptive cycle was initiated, starting from the effluent
phase.
[0042] FIG. 2: Result of a FISH image using the str probe (light
grey), specific for the Streptococcus genus (Trebesius et al. 2000)
compared to the EUB338 probe mix (dark grey) (Amann et al.
1990).
[0043] FIGS. 3a-b show microscopic images of bacterial cultures at
pH=5 (left) and pH=7 (right), clearly showing that different
bacteria are present at the higher pH.
[0044] FIG. 4: Development of the read abundance of the 16S rRNA
gene V3-V4 region in time on genus level. Various species are
found, which develop over time in amount. Streptococcus,
Enterobacteriaceae, Citrobacter, Klebsiella, and Clostridium are
found in relatively high amount.
Examples
[0045] Materials and Methods
[0046] Reactor Operation
[0047] The enrichment was carried out in a 3 L bioreactor with a
working volume of 2 L. Anaerobic conditions were maintained by
continuously sparging the reactor with N2 at a rate of 216 mL min-1
(@T=273K, P=105 kPa). The culture was taken out of the reactor for
biofilm removal from reactor walls and head three times per week.
The reactor was continuously agitated at a speed of 300 rpm using
mechanical stirrers. Reactor temperature was maintained at
30.quadrature.C by recirculating water heated at 31.degree. C.
(E300 thermostat, Lauda, Germany) in the outer wall of the reactor.
To prevent culture broth evaporation, the off-gas was cooled using
a cryostat set to 5.degree. C. Reactor pH was maintained at
5.0.+-.0.2 using 8 mol L.sup.-1 NaOH and 1 mol L.sup.-1 HCl
solutions (ADI 1030 Bio controller, Applikon, The Netherlands). To
prevent excessive foaming, anti-foaming agent (3% v:v anti-foam C,
Sigma Aldrich, Germany) was added in equal amounts and at equal
speed as NaOH during 10 g L.sup.-1 glucose fermentations.
[0048] Enrichment Medium
[0049] The enrichment at 10 g L.sup.-1 glucose was performed using
a medium to which NH.sub.4Cl, KH.sub.2PO.sub.4 and
FeCl.sub.2.4H.sub.2O were added to obtain a set molar C:N:P:Fe
ratio of 100:5:1:0.33 and 1.5 g yeast extract was added per 10 g of
glucose. Glucose and yeast extract solutions were autoclaved
separately at 110.degree. C. for 20 minutes and then combined.
Salts were supplied to the reactor from a second vessel, which was
autoclaved at 121.degree. C. Magnesium concentrations were adjusted
to increasing glucose concentrations. Trace elements were supplied
in sufficient amounts.
[0050] SBR Phases and Start-Up
[0051] The reactor was operated in SBR mode. In a start-up phase
for culture development the reactor was operated in batch mode
until glucose was entirely consumed. For inoculation of the
enrichment, 10 mL (0.5% v/v) of suspended and sieved (150 .mu.m
filtered) soil from the botanical garden of TU Delft was used (pH
7.4) and 10 mL of anaerobic digester sludge (Harnaschpolder, The
Netherlands). After the start-up phase the SBR mode with an
exchange ratio of 50% was entered. Three different SBR phases are
distinguished: the effluent phase (5 minutes), the feed phase (4
minutes) and the reaction phase. The length of the reaction phase
was dependent on the speed of microbial conversions in the reactor:
adaptive base adaptive cycling was used to control the adaptive
cycle time (FIG. 1). The base constant time was initially kept at
90 minutes to avoid biomass washout, as an initial lag phase was
observed in the adaptive cycles at the start of the enrichment.
Throughout the enrichment, the base constant time was gradually
decreased to 20 minutes.
[0052] Batch with 100 g L.sup.-1 Glucose at pH 5
[0053] Anaerobicity, temperature, pH, agitation and broth
evaporation were controlled as described for the sequencing batch
reactors. After 2.5 days of operation the reactor was spiked with
0.25 times the initial amount of trace elements.
[0054] Samples for monitoring biomass growth (OD660), glucose
consumption and product formation were taken every 30 minutes for
the first 2.5 hours of the cultivation. After this, samples were
taken every hour until t=8 h and finally 3 samples per day were
taken. The final biomass concentration was calculated by measuring
the volatile suspended solids (VSS) at the end of the
fermentation.
[0055] SBR Operation at pH 7
[0056] The enrichment at pH 7 was carried out as described for pH
5, but the pH was controlled at 7.2.+-.0.2 using 8 mol L.sup.-1
NaOH. The reactor was inoculated with 5 mL suspended and sieved
soil and 5 mL anaerobic digester sludge. Adaptive base adaptive
cycling with a minimal base constant time of 10 minutes was used
for the first two weeks of enrichment, after which a fixed adaptive
cycle length of 90 minutes was set. Initially, the culture broth
was agitated at 300 rpm. However, after 145 SRTs the stirring speed
was increased to 600 rpm to improve mass transfer of carbon dioxide
to the gas phase.
[0057] Batch with 100 g L.sup.-1 Glucose at pH 7
[0058] The batch reactor was operated as described for the batch
process at pH 5. The reactor was inoculated with cell pellets of
approximately 1 L of the culture obtained at the end of the
enrichment at pH 7. Samples were taken every 30 minutes for the
first 3.5 hours and every hour until glucose was nearly depleted
(<16 mM residual glucose). Anti-foam was added manually when
foaming occurred. Culture was stored in the fridge overnight before
collecting the pellet for VSS determination.
[0059] Analytical Methods
[0060] The gas productivities (H.sub.2 and CO.sub.2) and acid/base
dosages for pH control were monitored on-line using MFCS software
(Sartorius, Germany) and a Rosemount Analytical NGA 2000 (Emerson,
USA). Biomass concentrations were monitored both by measuring
optical density (OD660) and the amount of VSS in the broth as
described earlier using 150 mL effluent (APHA/AWWA/WEF 1999),
calculated assuming a biomass molecular weight of 24.6 g
mol.sup.-1. Both OD and VSS were always determined in duplicate.
Glucose, ethanol and VFA concentrations were determined using high
performance liquid chromatography (HPLC) using an Aminex HPX-87H
column (BioRad, USA) at 59.degree. C. coupled to a refractive
index- and ultraviolet detector (Waters, USA). 1.5 mmol L.sup.-1
phosphoric acid was used as eluent. Biomass was removed from the
reactor samples by centrifugation and filtration using a 0.22 .mu.m
membrane filter (Millipore, Millex-GV, Ireland).
[0061] Microbial Community Analysis
[0062] DNA was extracted from cell pellets from different time
points in the enrichment using the DNAeasy microbial extraction kit
(Qiagen Inc., Germany) following manufacturer's instructions and
sent to Novogene (Hong Kong, China) for 16S rRNA amplicon
sequencing of the V3-V4 region as described by Rombouts et al.,
(2019).
[0063] Fluorescent in situ hybridization (FISH) was used for
analyzing the microbial community with epifluorescence microscopy
(Axioplan 2 imaging, Zeiss, Germany). Fixation and overnight
hybridization were performed as described by Johnson et al. (2009)
using the probes listed in table 1. An additional DAPI staining
targeting all microbial cells was used by incubating the slides for
15 minutes with 10 .mu.L of 10 mg mL.sup.-1 DAPI solution after
washing and drying.
TABLE-US-00001 Forma- mide Sequence Microorganism Probe (%) (5'
.fwdarw. 3') Eubacteria EUB338 5-30 GCTGCCTCCCG TAGGAGT
Lactobacillus Lacto772 25 YCACCGCTACA CATGRAGTTCC ACT Lactococcus
Lactococcus4 5 CTGTATCCCGT GTCCCGAAG Megasphaera Mega-X 25
GACTCTGTTTT TGGGGTTT Streptococcus Str 30 CAC TCT CCC CTT CTG CAC
Enterobacteriaceae Ent183 20 CTC TTT GGT CTT GCG ACG
[0064] Results
[0065] A product spectrum was monitored in time of both
enrichments. Lactate was the main fermentation product in both
conditions. At 30 SRTs, the amount of trace elements was doubled,
leading to mainly lactate production. At pH 7, a 5.4 times higher
productivity was reached. What is very surprising is that only
L-lactic acid is produced at pH 7, while a nearly racemic mixture
is produced at pH 5.
TABLE-US-00002 pH 5 pH 7 10 g L.sup.-1 Maximum obtained lactate
yield 0.76 0.69 glucose (g.sub.p g.sub.s.sup.-1) Maximum obtained
productivity 1.16 6.24 (g L.sup.-1 h.sup.-) Ratio D:L-lactate 53:47
1:99 Y.sub.x/s (Cmol.sub.x Cmol.sub.s.sup.-1) 0.14 0.20 q.sub.s
(Cmol.sub.s Cmol.sub.x.sup.-1 h.sup.-1) 0.74 2.25 .mu..sub.average
(h.sup.-1) 0.11 0.46 100 g L.sup.-1 Y.sub.LA/s (g.sub.p
g.sub.s.sup.-1) 0.60 0.59 glucose Final attained lactate
concentration 57.6 56.6 (g L.sup.-1) 0.09 0.14 Y.sub.x/s
(Cmol.sub.xCmol.sub.s.sup.-1) 0.73 4.72 Average productivity (g
L.sup.-1 h.sup.-1)
[0066] The microbial community analysis revealed that the pH
enrichment was predominated by Lactobacillus species. A significant
side population of Megasphaera was detected.
[0067] At pH 7, Streptococcus was observed to be predominant genus,
with also several Enterobacteriaceae genera occurring, such as
Klebsiella and Citrobacter. The amount of Streptococcus was shown
to be very dominant, in the range of >90% of the biomass.
[0068] Further tests have been performed, using 100 g/l glucose
producing 82 g/l lactate, with a L:D ratio of 1:99. The biomass
yield was 0.08 Cmol-x/Cmol-S and a productivity of 2.35 g/L/h was
obtained.
CONCLUSIONS
[0069] Obtaining selective L-lactic acid production using mixed
cultures without sterilisation and a pure culture inoculum was
achieved. An acidic environment at pH 5 was tested in comparison to
a neutral pH environment at pH 7 under the same cultivation
conditions. A complex medium was used, as this stimulates the
growth of lactic acid bacteria. The adaptive cycle times could be
adjusted to the time base dosage stopped and the substrate,
glucose, was assumed to be depleted (the `plateau` was reached). It
was found that lactic acid bacteria could be successfully enriched
at both pH 5 and pH 7 using the described enrichment strategy.
Further, lactic acid production at pH 7 is favoured over pH 5, as
the productivity is higher and there is selective production of
L-lactic acid.
[0070] Inventors have obtained a high yield of lactic acid on
glucose using a defined medium and defined bioreactor conditions. A
stable yield of >70% LA g/g of glucose, and a productivity of
6.2 g/l*h in a 21 bioreactor at 10 g/l glucose and a titer of
lactic acid of 57 g/l is obtained in the reactor. These conditions,
or more explicitly, these ecological parameters, can be applied to
optimize current large-scale fermentations producing lactic acid.
Also, current waste streams can be used to ferment these to lactic
acid at high yield and refine the lactic acid out of the
stream.
[0071] With the present method and system, a maximum obtained yield
of 0.69 (molp mols.sup.-1), a maximum obtained productivity of 6.24
(g L.sup.-1 h.sup.-1), a ratio D:L-lactate of 0-100, and a final
attained lactate concentration of 56.6 (g L.sup.-1) were
obtained.
[0072] Below results of prior art documents and the present
invention are compared.
TABLE-US-00003 Table best achieved results on the basis of yield
Present Akao et Tang et al. Lang et al. Zhang et Parameter
invention al. 2007 2016 2015 al. 200 pH 7.0 6 pH to 6 every
Uncontrolled, 7 12 hours around 3.5 Temperature 30.degree. C.
55.degree. C. 37.degree. C. 35.degree. C. 35.degree. C. HRT and SRT
34 hours 240 hours 120 hours 24 hours 120 hours Ratio 7 31.4 10.9
56.1 25.2 peptides/carbohydrates (g/100 g) Carbohydrate
concentration 100 83 56.8 11.97 99.14 feed (g L.sup.-1) Yield
lactic acid on 0.82 0.73 0.54 0.63 0.65 carbohydrate (g/g)
Productivity (g LA L.sup.-1 h.sup.-1) 2.42 0.33 0.25 0.16 0.53
Lactic acid concentration at 82.3 39.6 30.5 7.5 64 the end of the
experiment, product titer (g L.sup.-1) Ratio D:L 1:99 3:97 Not
reported Not reported 40:60
[0073] So for the best results it is noted that Akaou used a
different temperature, with still lower yield and much lower
productivity, Tang sets the pH to 6 every 12 hours, which has
nothing to do with controlling, and Liang has an uncontrolled pH.
Yields of lactic acid are much lower, and productivity is a factor
lower, and typically almost an order lower. Also the D:L ratio is
much better.
* * * * *